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Dalton's theory was based on the concept that each element consists of its own unique brand of indivisible atom; atoms of one element are all alike but they differ from atoms of other elements. Importantly, Dalton assigned atomic weights to the atoms of the 20 elements he knew of at the time.

Dalton was born in Eaglesfield, Cumberland (now Cumbria), the son of a Quaker weaver. He became principal at a Quaker school in Kendal and taught there until 1793. He moved to Manchester to tutor in natural philosophy and science at the Manchester Academy, a Presbyterian college. However, his teaching duties left him with too little time to pursue his own scientific interests so he became a private tutor. One of his pupils was James Prescott Joule, later to become an influential physicist whose work led to the formulation of the first law of thermodynamics.

Dalton joined the Literary & Philosophical Society, which was at the centre of the scientific and business community in Manchester. The Society gave him a room for teaching and research at its premises on George Street. He read over 100 papers to the Society and became its Secretary, Vice-President and, ultimately, President.

Dalton was interested in the composition of the atmosphere and, by extension, in how components mix together to form gases. He formulated the Law of Partial Pressures in 1801, according to which the pressure of a mixed gas is the sum of the pressures that each of its components would exert if occupying the same space. He also developed the law of the thermal expansion of gases. Henry Roscoe, a later Manchester chemist, suggested that Dalton was trying to explain why the constituents of a gaseous mixture remain homogeneously mixed instead of separating into layers according to their density.

At the end of an 1803 paper on the absorption of gases by liquids, Dalton rather casually set out the first table of atomic weights. Encouraged by the favourable reception this paper received, he developed his theory further, in lectures to the Royal Society in 1803–1804 and later in his New System of Chemical Philosophy:

"...every particle of water is like every other particle of water; every particle of hydrogen is like every other particle of hydrogen...Chemical analysis and synthesis go no farther than to the separation of particles one from another, and to their reunion. No new creation or destruction of matter is within the reach of chemical agency."

This concept, that atoms of different elements were distinguished by differences in their weights, opened up new fields of experimentation. Each aspect of Dalton's theory has since been amended or refined, but its overall picture remains as the basis of modern chemistry and physics. Dalton also pioneered the use of ball-and-stick models to illustrate the three-dimensional structure of molecules.

Dalton also developed a theory to explain colour vision deficiency, from which he himself suffered. He suggested that the colour of the fluid in the eyes acted as a filter to certain colours in the spectrum. He left his eyes to be dissected after his death to test this theory; however, his theory was disproved. The DNA in his eyes was tested in the 1990s and showed the cause of his colour vision deficiency to be genetic.

John Dalton was widely honoured in his lifetime. He was elected one of the eight foreign associates of the French Academie des Sciences, a Fellow of the Royal Society and their first Royal Medallist. Oxford and Cambridge Universities both gave him honorary degrees. The City of Manchester commissioned his statue, which is in the Town Hall. At his death, 40,000 people filed past his coffin and there were 100 carriages in his funeral procession. Dalton is now regarded as a poor experimenter, but he had a powerful and vivid pictorial imagination that often gave him profound insights into the nature of atomic phenomena.